The goal of this proposal is to determine the role of the outer membrane spanning proteins or Oms of B. burgdorferi, sensu lato, in pathogenesis and immunity in Lyme disease. Oms and candidate Oms proteins have been identified in purified B. burgdorferi (Bb) outer membrane vesicles (OMV). Specific Aim 1 involves the characterization of Oms1 and Oms2, outer membrane spanning proteins with porin activity identified during the previous grant period. The porin, Oms1, is the first Bb protein which can be definitively termed an outer membrane spanning protein. Unlike other foreign porins, r-Oms1 is incorporated into the E. coli outer membrane, and exhibits native Oms1 conductance. Antibodies specific for r-Oms1 will be analyzed for their ability to bind the surface of Bb, to kill the spirochete in vitro, and to block spirochetal adhesion to host cells. The ability of r-Oms1 proteoliposomes to adhere to a variety of host cells will also be examined. Mice and rabbits immunized with r-Oms1 will be challenged intradermally with Bb, and potential protection determined. Oms2 is a second porin activity found in OMV. The impact of Oms2 on studies relating to protective immunity and pathogenesis will be assessed independently and in combination with Oms1. Additional candidate Oms proteins and their encoding genes will be characterized in Specific Aim 2. 19 hydrophobic, non-acylated proteins are found in OMV of virulent Bb strain B31, but not in OMV of avirulent ATCC strain B31. These proteins have been designated candidate Omsvsa, for virulent strain associated. Using virulent strain specific antiserum lambda EXlox phage clones expressing 12 distinct antigens have been identified; antigens of the same size have been found in virulent but not avirulent strain B31 and therefore represent candidate Omsvsa. Candidate Omsvsa shown to be bona fide Omsvsa will be further studies as for the porins, Oms1 and Oms2. The eppA gene found by Dr. Lovett and coworkers indicates that Bb has genes which are expressed during infection, but not during in vitro cultivation. Dr. Lovett hypothesizes that there may be Bb oms expressed only during infection, designated omsinf. In Specific Aim 3, strategies for recognition of candidate Omsinf genes and proteins are provided, and plans are also described for the determination of the roles of these genes and gene products in pathogenesis and immunity..